Multianalyte molecular analysis using application-specific random particle arrays

ABSTRACT

The present invention provides methods and apparatus for the application of a particle array in bioassay format to perform qualitative and/or quantitative molecular interaction analysis between two classes of molecules (an analyte and a binding agent). The methods and apparatus disclosed herein permit the determination of the presence or absence of association, the strength of association, and/or the rate of association and dissociation governing the binding interactions between the binding agents and the analyte molecules. The present invention is especially useful for performing multiplexed (parallel) assays for qualitative and/or quantitative analysis of binding interactions of a number of analyte molecules in a sample.

FIELD OF THE INVENTION

[0001] The present invention relates to mutiplexed bioassays foranalyzing binding interactions between analytes and binding agents,including methods for determining the affinity constants and kineticproperties associated with analyte-binding agent interactions.

BACKGROUND OF THE INVENTION

[0002] The imprinting of multiple binding agents such as antibodies andoligonucleotides on planar substrates in the form of spots or stripesfacilitates the simultaneous monitoring of multiple analytes such asantigens and DNA in parallel (“multiplexed”) binding assays. Theminiaturization of this array format for increasing assay throughput andstudying binding kinetics are described, for example, in R. Ekins, F. W.Chu, Clin. Chem. 37, 955-967 (1991); E. M. Southern, U. Maskos, J. K.Elder, Genomics 13, 1008-1017 (1992). In recent years, this approach hasattracted substantial interest particularly in connection withperforming extensive genetic analysis, as illustrated in G. Ramsay, Nat.Biotechnol. 16, 40-44 (1998); P. Brown, D. Botstein, Nat. Genet. 21,33-37 (1999); D. Duggan, M. Bittner, Y. Chen, P. Meltzer, J. M. Trent,Nat. Genet. 21, 10-14 (1999); R. Lipshutz, S. P. A. Fodor, T. R.Gingeras, D. J. Lockhart, Nat. Genet. 21, 20-24 (1999).

[0003] The principal techniques of array fabrication reported to dateinclude: refinements of the original “spotting” in the form of pintransfer or ink jet printing of small aliquots of probe solution ontovarious substrates, as illustrated in V. G. Cheung, et al., Nat. Genet.21, 15-19 (1999); sequential electrophoretic deposition of bindingagents in individually electrically addressable substrate regions, asillustrated in J. Cheng, et al., Nat. Biotechnol., 541-546 (1998); andmethods facilitating spatially resolved in-situ synthesis ofoligonucleotides, as illustrated in U. Maskos, E. M. Southern, NucleicAcids Res. 20, 1679-1684 (1992); S. P. A. Fodor, et al., Science 251,767-773 (1991) or copolymerization of oligonucleotides, as illustratedin A. V. Vasiliskov, et al., BioTechniques 27, 592-606 (1999). Thesetechniques produce spatially encoded arrays in which the position withinthe array indicates the chemical identity of any constituent probe.

[0004] The reproducible fabrication of customized arrays by thesetechniques requires the control of microfluidics and/or photochemicalmanipulations of considerable complexity to ensure consistentperformance in quantitative assays. Microfluidic spotting to produce, inquantitatively reproducible fashion, deposited features of 100 μmdiameter involves dispensing of nanoliter aliquots with tight volumecontrol, a task that exceeds the capabilities of currently availablefluid handling methodologies. In addition, exposure of binding agents toair during the deposition process, typically several hours' in duration,has uncontrollable impact on the molecular configuration and theaccessibility of the binding agents in subsequent binding assays.In-situ array synthesis relies on a sequence of multiple masking andphotochemical reaction steps which must be redesigned to accommodate anychanges in array composition. Finally, assay performance must beassessed “in-situ” for each array subsequent to immobilization ofbinding agents, an aspect of array manufacturing which raises difficultquality control and implementation issues.

SUMMARY OF THE INVENTION

[0005] The present invention provides methods and apparatus for theapplication of a particle array in bioassay format to performqualitative and/or quantitative molecular interaction analysis betweentwo classes of molecules (an analyte and a binding agent). The methodsand apparatus disclosed herein permit the determination of the presenceor absence of association, the strength of association, and/or the rateof association and dissociation governing the binding interactionsbetween the binding agents and the analyte molecules. The presentinvention is especially useful for performing multiplexed (parallel)assays for qualitative and/or quantitative analysis of bindinginteractions.

[0006] The terms “analyte” and “binding agent” refer to moleculesinvolved in binding interactions. In one example, analyte and bindingagent include DNA or RNA fragments (e.g., oligonucleotide), and bindingof those fragments to their complementary sequences (hybridization) isanalyzed. In another example, binding interactions between ligands andreceptors are analyzed. Examples of analytes and binding agents alsoinclude aptamers, peptides and proteins (e.g., antibodies), antigens,and small organic molecules.

[0007] The term “particles” refer to colloidal particles, includingbeaded polymer resins (“beads”).

[0008] The present invention also provides automated, on-demandfabrication of planar arrays composed of a selected mixture ofchemically distinct beads (e.g., encoded beads) which are disposed on asubstrate surface in accordance with a selected spatial configuration,as described above. In this approach, the beads are functionized todisplay binding agents. For example, the binding agents may be attachedto the beads, preferably by covalent bond. The subsequent qualitycontrol and performance evaluation are conducted off-line and areindependent from the process of array assembly. The separation of stepssuch as bead encoding, functionalization and testing; substrate design,processing and evaluation; custom assembly of application-specificarrays; and on-line decoding of arrays enable an otherwise elusivecombination of flexibility, reliability and low cost by permittingsystematic process control.

[0009] The methods disclosed herein permit rapid customization of DNA orprotein arrays without the need for process redesign and avoid problemscontributing to spot-to-spot as well as chip-to-chip variability.Furthermore, the bead array format permits chip-independentcharacterization of beads as well as optimization of assay conditions.In addition, multiple bead arrays can be formed simultaneously indiscrete fluid compartments maintained on the same chip, permitting theconcurrent processing of multiple samples.

BRIEF DESCRIPTION OF DRAWINGS

[0010] Other objects, features and advantages of the invention discussedin the above brief explanation will be more clearly understood whentaken together with the following detailed description of an embodimentwhich will be understood as being illustrative only, and theaccompanying drawings reflecting aspects of that embodiment, in which:

[0011]FIG. 1 is an illlustration of process flow including theproduction of random encoded bead arrays and their use in multiplexedassays

[0012]FIG. 2 is an Illustration of the functionalization of beads

[0013]FIG. 3 is an illustration of steps in chip design and wafer-scaleproduction

[0014]FIG. 4 is an illlustration of on-demand assembly of random encodedarrays

[0015]FIG. 5 is an illustration of palmtop microlab

[0016]FIG. 6 is a schematic illustration of assay and decoding imagesused in READ process

[0017]FIG. 7 is a flow chart summarizing algorithms and steps in theanalysis of images

[0018]FIG. 8 is an illustration of steps in the decomposition of assayimages according to bead type by application of the image analysisalgorithm summarized in FIG. 7.

[0019]FIG. 9 is an illustration of optically programmable array assemblyof random encoded arrays

[0020]FIG. 10 is an illustration of an array composed of random encodedsubarrays

[0021]FIG. 11 is an illustration of stations in an automated chip-scalebead array manufacturing and QC process

[0022]FIG. 12 is an illustration of quantitative binding curves for twocytokines

[0023]FIG. 13 is an illustration of array design for polymorphismanalysis

[0024]FIG. 14 is a fluorescence micrograph of assay and decoding imagesrecorded from one subarray shown in FIG. 13 in the course ofpolymorphism analysis

[0025]FIG. 15 is an illustration of assay results in the form ofintensity histograms obtained from the analysis of assay images such asthe one illustrated in FIG. 14.

[0026]FIG. 16 is an illustration of design of a “looped probe” forhybridization assays

[0027]FIGS. 17A and 17B are fluorescence micrographs of assay anddecoding images recorded in the course of the analysis of multiplecytokines

[0028]FIGS. 18A and 18B are illustrations of numerical simulations ofcross-correlations in receptor-ligand systems with multiple competingreceptor-ligand interactions

[0029]FIG. 19 is an illustration of numerical simulations ofreceptor-ligand association and disassociation kinetics

[0030]FIG. 20 is an illustration of integrated sample capture usingmagnetic capture beads and array-based detection using READ

[0031]FIG. 21 is an illustration of multi-step assays using encodedmagnetic beads to integrate gene-specific capture, on-bead reversetranscription and post-assay array assembly

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The fabrication of application-specific bead arrays may involvemultiple processes in a multi-step sequence which may be automated usingexisting liquid handling technology and laboratory automation. Theprocess of Random Encoded Array Detection (READ) includes thefabrication of custom bead arrays as well as the use of such arrays inbioassays, including assays involving multiplexed molecular interactionanalysis of analyte and binding agent molecules, including DNA andprotein analysis.

[0033]FIG. 1 provides a schematic overview of the functional componentsand process flow by which custom bead arrays may be prepared and used inperforming multiplexed biomolecular analysis according to the presentinvention. The array is prepared by employing separate batch processesto produce application-specific substrates (e.g., chip at the waferscale) and to produce beads that are chemically encoded and biologicallyfunctionalized (e.g., at the scale of ˜10{circumflex over ( )}8beads/100 μl of suspension). Tthe beads subjected to respective qualitycontrol (QC) steps prior to array assembly, such as the determination ofmorphological and electrical characteristics. In addition, actual assaysare performed on beads in suspension, before they are introduced to thesubstrate, to optimize assay conditions, generally with the objective tomaximize assay sensitivity and specificity and to minimize bead-to-beadvariations. For substrates, QC steps may include optical inspection,ellipsometry and electrical transport measurements.

[0034] Once the chemically encoded and biologically functionalized beadsare combined with the substrate (e.g., chip), the Light-controlledElectrokinetic Assembly of Particles near Surfaces (LEAPS) may be usedfor rapid assembly of dense arrays on a designated area on the substratewithin the same fluidic phase, avoiding problems contributing tospot-to-spot as well as chip-to-chip variability without the need forretooling or process redesign. Furthermore, the bead array formatpermits chip-independent characterization of beads as well asoptimization of assay conditions. In addition, multiple bead arrays canbe formed simultaneously in discrete fluid compartments maintained onthe same chip. Once formed, these multiple bead arrays may be used forconcurrent processing of multiple samples. The integration of LEAPS withmicrofluidics produces a self-contained, miniaturized, opticallyprogrammable platform for parallel protein and DNA analysis. LEAPSrefers to methods of moving particles suspended in the interface betweenan electrolyte solution and an electrode and is described in U.S. patentapplication Ser. No. 09/171,550 (also PCT International Application No.PCT/US97/08159) entitled Light-controlled Electrokinetic Assembly ofParticles near Surfaces, which is incorporated herein by reference inits entirety. Also incorporated herein by reference in its entirety isU.S. patent applications Ser. No. 09/397,793 (also PCT InternationalApplication PCT/US00/25466), entitled “System and Method forProgrammable Pattern Generation.

[0035] In certain embodiments of the present invention, chemicalencoding may be accomplished by staining beads with sets of opticallydistinguishable tags, such as those containing one or more fluorophoredyes spectrally distinguishable by excitation wavelength, emissionwavelength, excited-state lifetime or emission intensity. The opticallydistinguishable tags made be used to stain beads in specified ratios, asdisclosed, for example, in Fulwyler, U.S. Pat. No. 4,717,655 (Jan. 5,1988). Staining may also be accomplished by swelling of particles inaccordance with methods known to those skilled in the art, [Molday,Dreyer, Rembaum & Yen, J. Mol Biol 64, 75-88 (1975); L. Bangs, “Uniformlatex Particles, Seragen Diagnostics, 1984]. For example, up to twelvetypes of beads were encoded by swelling and bulk staining with twocolors, each individually in four intensity levels, and mixed in fournominal molar ratios. Combinatorial color codes for exterior andinterior surfaces is disclosed in International Application No. PCT/US98/10719, which is incorporated herein by reference in its entirety.

[0036] Beads are functionalized by binding agent molecules attachedthereto, the molecule including DNA (oligonucleotides) or RNA fragments,peptides or proteins, aptamers and small organic molecules in accordanceprocesses known in the art, e.g., with one of several coupling reactionsof the known art (G. T. Hermanson, Bioconjugate Techniques (AcademicPress, 1996); L. Illum, P. D. E. Jones, Methods in Enzymology 112, 67-84(1985). In certain embodiments of the invention, the functionalizedbeads have binding agent molecules (e.g., DNA, RNA or protein)covalently bonded to the beads. Beads may be stored in a buffered bulksuspension until needed. Functionalization typically requires one-stepor two-step reactions which may be performed in parallel using standardliquid handling robotics and a 96-well format to covalently attach anyof a number of desirable functionalities to designated beads, asillustrated in FIG. 2. In a preferred embodiment, beads of core-shellarchitecture will be used, the shell composed in the form of a thinpolymeric blocking layer whose preferred composition is selected; andfunctionalization performed in accordance with the targeted assayapplication, as known in the art. Samples may be drawn for automated QCmeasurements. Each batch of beads provides material for hundreds ofthousands of chips so that chip-to-chip variations are minimized.

[0037] Substrates (e.g., chips) used in the present invention may bepatterned in accordance with the interfacial patterning methods of LEAPSby, e.g., patterned growth of oxide or other dielectric materials tocreate a desired configuration of impedance gradients in the presence ofan applied AC electric field. Patterns may be designed so as to producea desired configuration of AC field-induced fluid flow and correspondingparticle transport. Substrates may be patterned on a wafer scale byinvoking semiconductor processing technology, as illustrated in FIG. 3.In addition, substrates may be compartmentalized by depositing a thinfilm of a UV-patternable, optically transparent polymer to affix to thesubstrate a desired layout of fluidic conduits and compartments toconfine fluid in one or several discrete compartments, therebyaccomodating multiple samples on a given substrate.

[0038] In certain embodiments of the invention, the bead array isprepared by providing a first planar electrode that is in substantiallyparallel to a second planar electrode (“sandwich” configuration) withthe two electrodes being separated by a gap and containing anelectrolyte solution. The surface or the interior of the second planarelectrode is patterned with the interfacial patterning method. Encodedand functionalized beads are introduced into the gap. When an AC voltageis applied to the gap, the beads form a random encoded array on thesecond electrode (e.g., “chip”). And, also using LEAPS, an array ofbeads may be formed on a light-sensitive electrode (“chip”). Preferably,the sandwich configuration described above is also used with a planarlight sensitive electrode and another planar electrode. Once again, thetwo electrodes are separated by the a gap and contain an electrolytesolution. The functionalized and encoded beads are introduced into thegap. Upon application of an AC voltage in combination with a light, thebeads form an array on the light-sensitive electrode.

[0039] In certain embodiments, the application-specific bead arraysuseful in the present invention may be produced by picking aliquots ofdesignated encoded beads from individual reservoirs in accordance withthe specified array composition and “pooled”; aliquots of pooledsuspension are dispensed onto selected substrate (e.g., chips) in amanner preventing the initial fusion of aliquots. Aliquots form amultiplicity of planar random subarrays of encoded beads, each subarrayrepresenting beads drawn from a distinct pool and the physical arraylayout uniquely corresponding to the identity of aliquots drawn frompooled bead populations.

[0040] Planar arrays or assemblies of encoded on a substrate which arechemically or physically encoded may be used. To this, spatial encodingmay also be added to increase the number of assays that may beconducted. Spatial encoding, for example, can be accomplished within asingle fluid phase in the course of array assembly by invokingLight-controlled Electrokinetic Assembly of Particles near Surfaces(LEAPS) to assemble planar bead arrays in any desired configuration inresponse to alternating electric fields and/or in accordance withpatterns of light projected onto the substrate. LEAPS creates lateralgradients in the impedance of the interface between silicon chip andsolution to modulate the electrohydrodynamic forces that mediate arrayassembly. Electrical requirements are modest: low AC voltages oftypically less than 10V_(pp) are applied across a fluid gap of typically100 μm between two planar electrodes. This assembly process is rapid andit is optically programmable: arrays containing thousands of beads areformed within seconds under electric field. The formation of multiplesubarrays, can also occur in multiple fluid phases maintained on acompartmentalized chip surface.

[0041] The multiplexed assays of the present invention may also beperformed using beads encoded beads that are assembled, but not in anarray, on the substrate surface. For example, by spotting beadsuspensions into multiple regions of the substrate and allowing beads tosettle under gravity, assemblies of beads can be formed on thesubstrate. In contrast to the bead arrays formed by LEAPS, theseassemblies generally assume low-density, disorder configurations.However, the combination of spatial and color encoding attained byspotting mixtures of chemically encoded beads into a multiplicity ofdiscrete positions on the substrate still provides a degree ofmultiplexing that is sufficient for certain biological assays.

[0042] Binding interaction between the binding agent on those beads andan analyte may be performed either before or after the encoded array isassembled on the substrate. For example, the bead array may be formedafter the assay, subsequent to which an assay image and a decoding imagemay be taken of the array. Alternatively, the beads may be assembled inan array and immobilized by physical or chemical means to produce randomencoded arrays, e.g., with the appearance of the array shown in FIG. 10.The arrays may be immobilized, for example, by application of a DCvoltage to produce random encoded arrays with the appearance of thearray shown in FIG. 10. The DC voltage, set to typically 5-7 V (forbeads in the range of 2-6 μm and for a gap size of 100-150 μm) andapplied for <30s in “reverse bias” configuration so that an n-dopedsilicon substrate would form the anode, causes the array to becompressed to an extent facilitating contact between adjacent beadswithin the array and simultaneously causes beads to be moved toward theregion of high electric field in immediate proximity of the electrodesurface. Once in sufficiently close proximity, beads are anchored by vander Waals forces mediating physical adsorption. This adsorption processis facilitated by providing on the bead surface a population of“tethers” extending from the bead surface; polylysine and streptavidinhave been used for this purpose.

[0043] In certain embodiments, the particle arrays may be immobilized bychemical means, e.g, by forming a composite gel-particle film. In oneexemplary method for forming such gel-composite particle films, asuspension of microparticles is provided which also contain allingredients for subsequent in-situ gel formation, namely monomer,crosslinker and initiator. The particles are assembled into a planarassembly on a substrate by application of LEAPS, e.g., AC voltages of1-20 V_(p-p) in a frequency range from 1 00's of hertz to severalkilohertz are applied between the electrodes across the fluid gap.Following array assembly, and in the presence of the applied AC voltage,polymerization of the fluid phase is triggered by thermally heating thecell ˜40-45° C. using an IR lamp or photometrically using a mercury lampsource, to effectively entrap the particle array within a gel. Gels maybe composed of a mixture of acrylamide and bisacrylamide of varyingmonomer concentrations from 20% to 5% (acrylamide:bisacrylamide=37.5:1,molar ratio), or any other low viscosity water soluble monomer ormonomer mixture may be used as well. Chemically immobilizedfunctionalized microparticle arrays prepared by this process may be usedfor a variety of bioassays, e.g., ligand receptor binding assays.

[0044] In one example, thermal hydrogels are formed usingazodiisobutyramidine dihydrochloride as a thermal initiator at a lowconcentration ensuring that the overall ionic strength of thepolymerization mixture falls in the range of ˜0.1 mM to 1.0 mM. Theinitiator used for the UV polymerization is Irgacure 2959®(2-Hydroxy-4′-hydroxyethoxy-2-methylpropiophenone, Ciba Geigy,Tarrytown, N.Y.). The initiator is added to the monomer to give a 1.5%by weight solution.

[0045] In certain embodiments, the particle arrays may be immobilized bymechanical means. For example, an array of microwells may be produced bystandard semiconductor processing methods in the low impedance regionsof the silicon substrate. The particle arrays may be formed using suchstructures by, e.g., utilizing LEAPS mediated hydrodynamic andponderomotive forces are utilized to transport and accumulate particleson the hole arrays. The A.C. field is then switched off and particlesare trapped into microwells and thus mechanically confined. Excess beadsare removed leaving behind a geometrically ordered random bead array onthe substrate surface.

[0046] When the bead array is immobilized before the assay, the arrayfunctions as a two-dimensional affinity matrix which displays receptorsor binding agents (e.g., oligonucleotides, cDNA, aptamers, antibodies orother proteins) to capture analytes or ligands (DNA, proteins or othersmall cognate ligands) from a solution that is brought in contact withthe array. The bead array platform may be used to perform multiplexedmolecular analysis, such as, e.g., genotyping, gene expressionprofiling, profiling of circulation protein levels and multiplexedkinetic studies.

[0047] Substrates (e.g., chips) can be placed in one or more enclosedcompartment, permitting samples and reagents to be transported in andout of the compartments through fluidic interconnection. On-chipimmunoassays for cytokines, e.g., interleukin (IL-6) may be performed inthis format. Serum sample and fluorescent labeled secondary antibodiesare introduced to the reaction chamber sequentially and allowed to reactwith beads immobilized on the chip. FIG. 5 illustrates a design of areaction chamber which may be used in the multiplexed assays accordingto the present invention. Reactions can also be performed in an opencompartment format similar to microtiter plates. Reagents may bepipetted on top of the chip by robotic liquid handling equipment, andmultiple samples may be processed simultaneously. Such a formataccommodates standard sample processing and liquid handling for existingmicrotiter plate format and integrates sample processing and arraydetection.

[0048] In certain embodiments, the presence of the analyte-binding agentinteractions are associated with changes in the optical signatures ofbeads involved in the interactions and these optical changes detectedand analyzed. The identities of the binding agents involved in theinteractions are determined by detecting the chemically or physicallydistinguishable characteristic associated with those beads. Preferably,chemically distinguishable characteristics include chemical moleculesincluding flurophore dyes, chromophores and other chemical moleculesthat are used for purposes of detection in binding assays.

[0049] The detection of the chemically or physically distinguishablecharacteristic and the detecting of the optical signature changesassociated with the binding interactions may be performed while theparticles are assembled in a planar array on a substrate, e.g., bytaking an assay and a decoding image of the array and comparing the two,e.g., comparing of the assay and the decoding image comprises use ofoptical microscopy apparatus including an imaging detector andcomputerized image capture and analysis apparatus. The decoding imagemay be taken to determine the chemically and/or physicallydistinguishable characteristic that uniquely identifies the bindingagent displayed on the bead surface, e.g., determining the identity ofthe binding agents on each particle in the array by the distinguishablecharacteristic. The assay image of the array is taken to detect theoptical signature of the binding agent and the analyte complex. Incertain embodiments, fluorescent tags (fluorophore dyes) may be attachedto the analytes such that when the analytes are bound to the beads, theflourescent intensities change, thus providing changes in the opticalsignatures of the beads. In certain embodiments, tthe decoding image istaken after the beads are assembled in an array and immobilized andbefore taking the assay image, preferably before contacting the bindingagents on the beads with an analyte. In certain other examples, thebinding interactions occur while the beads are in solution, andassembled into an array afterwards and the decoding and assay images areobtained. The identity of the binding agent of the binding agent-analytecomplex is carried out by comparing the decoding image with the assayimage.

[0050] In preferred embodiments, images analysis algorithms that areuseful in analyzing the data obtained from the decoding and the assayimages. These algorithm may be used to obtain quantitative data for eachbead within an array. As summarized in FIG. 7, the analysis softwareautomatically locates bead centers using a bright-field image of thearray as a template, groups beads according to type, assignsquantitative intensities to individual beads, rejects “blemishes” suchas those produced by “matrix” materials of irregular shape in serumsamples, analyzes background intensity statistics and evaluates thebackground-corrected mean intensities for all bead types along with thecorresponding variances.

[0051] The methods of the present invention may be used for determiningthe association and the dissociation constants e.g., by introducing theanalyte in a time-dependent manner and analyzing the binding as afunction of time, or by washing away the bound analyte in atime-dependent manner and also analyzing the binding as a function oftime.

[0052] The methods of the present invention may be used for determiningthe affinity constants of analyte-binding agent interactions, fordetermining the number of analyte-binding agent complexes formed

[0053] The present invention also provides methods for determining theconcentration of an analyte in a biological sample.

[0054] The methods of the present invention may also be used todetermining elements of a co-affinity matrix of a given analyte againsta panel of binding agents. In one example, the extent of the interactionbetween the analyte and the binding agents in a panel in competitive,multiconstituent equilibrium reaction may be determined. Determinationof co-affinity constants provides useful applications, as describedbelow.

[0055] The successful rate of transplantation for several types oforgans directly relates to compatibility of Human Leukocyte Antigen(HLA) between donor and recipient. Serological testing of the recipientsfor the Panel Reactive Antibodies (PRA) is one of the crucial steps toavoid possible rejections. Cross-reaction in PRA testing is a verycommon phenomenon due to similarity of some HLA antigen structures andthe nature of development of these antibodies. In fact, HLA antigens canbe organized into groups based on apparent serological cross-reactivitybetween the groups. These groups are termed Cross-Reactive-Groups(CREGs). In current clinical setting, antibodies from a patient aretested against different antigens in individual reactions. Although areactive pattern of the antibodies can be generated combining theresults from different reactions, the competitive nature of interactionsbetween different antibodies and antigens is not reflected in such apattern. In other cases, several antigens are mixed together for abinding assay. Lack of identification of each antigen in the systemprevents generation of a binding profile. The result is only theaveraged signal from several antigens. In the bead array system, a panelof different antigens is presented to the antibody analytes in acompetitive binding environment, and each antigen can be identifiedthrough its association with different types of beads. Thus, bindingintensity on each antigen in the competitive reactions can be extractedin a single assay. This co-affinity matrix system will provide bindingprofiles for the CREGs and greatly advance the understanding of thenature of the reaction and improve the accuracy for the related clinicaldecisions. For example, a N-antibody and M-antigen system provides amatrix of N×M of possible reactions. It is possible to determine K−nm,the affinity constant governing the interaction between the nth antibodyagainst the mth antigen, where m=1, 2, . . . M, and n=1, 2, . . . N. Forapplications where absolute co-affinity constants are not needed,binding profile will be generated for various antibodies in accordancewith the methods of the present invention and results from a patientsample can be matched to these profiles or combination of theseprofiles.

[0056] Co-affinity matrix may also be used to characterize the analyte.For example, combination of the coefficients of the co-affinity matrixand known concentrations of analyte and binding agents participating inthe formation of analyte-binding agent complexes serves to define acompetitive binding interaction descriptor, e.g.,The molecularinteraction parameter,${P_{n}\left( R_{m} \right)} = \frac{K_{mn}\left\lbrack L_{n} \right\rbrack}{\sum\limits_{j}^{\quad}\quad {K_{mj}\left\lbrack L_{j} \right\rbrack}}$

[0057] provides a characterization of the molecular interaction betweena binding agent, R_(m), and an analyte, L_(n), in the presence ofanalytes {L_(j); 1≦j≦N}, all of which exhibit a finite affinity, K_(mj),for that binding agent. That is, P_(n), 0≦P_(n)≦1, represents anormalized specificity of binding agent R_(m) for analyte L_(n) in amulticonstitutent competitive reaction and serves as a robustcharacterization of that binding agent based on co-affinities displayedin a multiconstituent competitive reaction. See also P. H. von Hippel etal., Proc. Natl. Acad. Sci. USA 83, 1603 (1986), incorporated herein byreference.

[0058] The pattern of binding interaction of a analyte against a panelof binding agents may be used to characterize the analyte and compare itwith other molecules. In addition, by generating the co-affinity matrixof a analyte using a reference panel of binding agents, such affinitymay be used to determine if a sample later introduced to the panel ofbinding agents contains an impurity by observing the deviation in thebinding pattern.

[0059] The present invention also provides use of superparamagneticparticles (“magnetic particles”) as described in U.S. Pat. No. 5,759,820and European Patent No. 83901406.5 (Sintef), which may then be used inintegrated the sample preparation step with the assay step involvingencoded bead arrays. Both of these references are incorporated herein byreference.

[0060] Superparamagnetic particles may be encoded with a chemically orphysically distinguishable characteristic (e.g., flourescent tag) andused performing bioassays of the present invention. In certainembodiments, the particles are assembled using LEAPS, as withnon-magnetic encoded beads. The encoded also be used in arraygeneration, and assayed. The present invention also includes theformation of a planar array of encoded and functionalizedsuperparamagnetic particles on a substrate by application of magneticfield to said particles.

[0061] Several methods for the synthesis of monodispersesuperparamagnetic microspheres are known in the art. G. Helgesen et al.,Phys. Rev. Lett. 61, 1736 (1988), for example, disclosed a method whichutilizes porous and highly cross-linked polystyrene core particles whoseinterior surfaces are first nitrated, following which iron oxides areprecipitated throughout the particle to produce a paramagnetic core.Following completion of this step, the particles are coated withfunctional polymers to provide a reactive shell. U.S. Pat. No. 5,395,688to Wang et al. describes a process for producing magnetically responsivefluorescent polymer particles composed of a fluorescent polymer coreparticle that is evenly coated with a layer of magnetically responsivemetal oxide. The method utilizes preformed fluorescent polymeric coreparticles which are mixed with an emulsion of styrene and magnetic metaloxide in water and polymerized. A two step reactive process such as thissuffers from the drawback of possible inhibition of polymerization bythe fluorescent dye or conversely bleaching of the fluorescence by theshell polymerization process.

[0062] The method also provides a novel process for making color encodedmagnetic beads, a simple and flexible one-step process to introduce intopreformed polymeric microparticles a well controlled amount of magneticnanoparticles, prepared in accordance with the procedure describedbelow, along with well controlled quantities of one or more fluorescentdyes. In an embodiment of the present invention, the quantity of themagnetic nanoparticles. is controlled to produce magnetic particles thatform an array on a substrate upon application of magnetic field to saidparticles. This process involves swelling the polymer particles in anorganic solvent containing dyes and magnetic nanoparticles and thereforeapplies to any polymer particle which can be subjected to standardswelling procedures such as those disclosed in the prior art offluorescent staining of microparticles. Unlike encoding methods in whichthe magnetic material and the fluorescent dyes are each located todifferent areas of the (core/shell) of the magnetic particle, uniformswelling of particles ensures the distribution of magnetic particlesthroughout the interior volume. This process also permits thequantitative control of the nanoparticle as well as dye content over awide range, thereby permitting the tailoring of the particles' magneticsusceptibility as well as fluorescence intensities. An additional methodof the present invention to control the magnetic properties of the hostparticles, other than to control loading, is to tune the size of themagnetic nanoparticles by adjusting the water content of the micellarsynthesis reaction (see below).

[0063] Physical or chemical coupling of biomolecules possible on theparticle surface utilizing preexisting functional groups. Leaching outof magnetic nanoparticles is readily eliminated by growing a furtherpolymeric shell on the particle.

[0064] In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes and are notto be construed as limiting this invention in any manner.

EXAMPLES Example 1 Optically Programmable Array Formation

[0065] As illustrated in FIG. 9, LEAPS serves to simultaneously assemblemultiple random encoded subarrays and to “drag-and-drop” these subarraysinto separate, but proximate locations on the chip within a common,enclosed liquid environment. Two sets of beads (2.8 μm Oligo-(dT)₂₅,Dynal, Oslo, Norway), dispensed from separate reservoirs A and B, weresimultaneously assembled into distinct subarrays within the same fluid;sub-arrays were then simultaneously placed into desired destinations asdirected by spatially varying illumination profiles which were generatedand projected onto the substrate by a PC-programmable illuminationpattern generator (described in U.S. Ser. No. 09/397,793, filed Sep. 17,1999, which is incorporated herein by reference in its entirety). Thisdrag-and-drop operation reduced the separation between the twosub-arrays from approximately 250 μm to 20 μm. Beads were moved at 5V_(pp) at a frequency of 2 kHz; total power projected onto the substratesurface was ˜5 mW. The combination of chemical and spatial encodingpermits a given set of chemical bead markers to be used multiple timesand reduces the demands placed on either encoding dimension whilefacilitating the realization of large coding capacities.

Example 2 Array Formation on Patterned Surface

[0066] Illustrated in FIG. 10 is an array of encoded beads assembled ona patterned silicon chip using an AC voltage of 1-2 V_(pp) and afrequency of 100-150 Hz, applied across a 100 μm electrode gap filledwith an aqueous bead suspension; a thermal oxide (˜1000 Å) on thesubstrate was patterned by etching the oxide to a thickness of 50-100 Åin a set of square features (˜30×30 μm²) on 130 μm centers; arrays ofsimilar layout also can be produced in response to suitable illuminationpatterns. Each sub-array shown here contains approximately 80 beadscoupled with anti-cytokine monoclonal antibodies. Carboxylate-modifiedpolystyrene beads of 5.5 μm diameter (Bangs Laboratory, Fishers, Ind.)were stained with a combination of two types of fluorescent dyes andwere then functionalized with anti-cytokine-mAb. The assembly processensures collection of all beads at the substrate surface. Bead encodingwas as follows: IL-2 (Bright Red); IL-4 (Dim Red); IL-6 (Bright Green);M-CSF (Dim Green) and TNF-α (Yellow).

Example 3 Formation of Arrays of Magnetic Particles

[0067] Colloidal particles exhibiting a finite diamagneticsusceptibility, when disposed on a planar substrate can be assembledinto ordered arrays in response to increasing magnetic fields.Commercially available superparamagnetic particles (Dynal, Oslo, NO),dispersed from a fluid suspension onto the planar surface of the lowerof two parallel bounding surfaces of a fluid cell (“sandwich” geometry),when exposed to a homogeneous axial magnetic field (oriented normal tothe substrate plane), will form ordered assemblies. As a function ofincreasing magnetic field strength, and for given diamagneticsusceptibility of the particles as controlled by the manufacturingprocess known to the art, ordered planar assemblies and linear stringsof beads oriented normal to the substrate can be formed. Permanentmagnets can be designed so as to produce the field strength required torealize the desired configuration of the assembly. Requisite magneticfield configurations can be produced by an electromagnet in solenoid orHelmholtz configuration known to the art; the substrate can beintroduced into the magnet bore or can be placed in immediate proximityto the coil(s) outside of the bore so as to ensure the orientation ofthe field substantially normal to the substrate plane. Spatiallymodulated magnetic fields can be produced by patterning the substratewith permalloy using methods known to the art.

Example 4 Formation of Random Bead Assemblies

[0068] Aliquots of solution containing suspended beads were placed ontoseveral distinct positions on a planer substrate of silicon capped witha thin silicon oxide layer (other substrates may be used here). Beadswere allowed to settle under gravity to form random assemblies. Todelineate discrete positions on the substrate, one of the following twomethods were used. According to the first method, a silicon gasket (of250 um thickness), displaying a grid of multiple round holes of 1 mm or2 mm diameter (Grace Bio-labs, Bend, Oreg.) is placed on thehydrophillic surface to define microwells (of 0.25 to 0.5 ul volume) formultiple discrete samples of bead suspension. According to the secondmethod, small aliquots of fluid containing beads (0.2 ul to 0.5 ul involume) are directly placed onto a hydrophillic surface in one or moredesignated areas so as to ensure formation of discrete droplets; spacersare not needed in this case. As solvent evaporates (at room temperatureor, for rapid drying, at elevated temperature ( about 60 C), beads areleft in random positions on the substrate. DNA polymorphism reactionshave been tested in assemblies formed in both manners. Optionally, beadssettling under gravity may be immobilized by chemical capture layersprovided on the substrate. An application of random bead assemblies todetermine affinity constants in a multiplexed format is described inExample 6.

Example 5 An Automated Chip-scale Array Manufacturing Process

[0069] As illustrated in FIG. 11, the process involves liquid handlingand pipetting of beads onto chips mounted in single-chip cartridges ormulti-chip cartridges. Bead arrays are formed using methods such asthose in Examples 1, 2 or 3., followed by array immobilization anddecoding. The resulting decoding images are stored for later use alongwith an optional chip ID (“bar code”).

Example 6 Determination of Affinity Constants by Post-assay Analysis ofBead Assemblies

[0070] Quantitative binding curves for the cytokines TNF-α and IL-6.Binding curves were generated by performing sandwich immunoassays usingchemically encoded beads in suspension, said suspensions being confinedto one or more reaction compartments delineated on-chip, or in one ormore reaction compartments off chip. By completing the reaction withbeads maintained in suspension, assay kinetics similar to homogeneousassays can be attained. Following completion of the binding reaction,beads were assembled on chip to permit multiplexed quantitative imageanalysis. Random assemblies prepared according to Example 4 or orderedbead arrays prepared according to Example 1 or 2 may be used. Anadvantage of ordered, dense assemblies produced by the methods ofExamples 1 or 2 is the higher spatial density and higher assaythroughput attained by processing a greater number of beads.

[0071] As an illustration, FIG. 12 displays quantitative binding curvesfor TNF-α and IL-6, obtained from randomly dispersed beads. Acommercial-grade 8-bit video-CCD camera (Cohu, San Diego, Calif.) wasused in a mode permitting multi-frame integration. The range ofconcentrations of antigen used in the two assays was 700 fM to 50 pM forTNF-α and 2 pM to 50 pM for IL-6. At each concentration, the number ofmolecules bound per bead was estimated by comparison with calibrationbeads coated with known quantities of Cy5.5-labeled BSA per bead;requisite adjustments were made to account for differences influorescence quantum efficiency between labeled secondary antibodies andBSA.

[0072] This format of analysis permits the determination of the affinityconstant, K_(A)=[LR]/([R₀-LR][L]), where, in accordance with the law ofmass action, [LR] denotes the number of receptor-ligand pairs per beadand [L] denotes the solution concentration of ligand. By specifying thenumber of beads per ml, n_(B), and specifying a value for [R₀] in termsof the number of receptors per bead, theoretical binding curves,computed for given K_(A), are compared to a plot of the number of boundmolecules per bead as a function of bulk ligand concentration. Theabsolute number of ligands bound per bead may be determined for givenbulk concentration by measuring the mean fluorescence intensity per beadand referencing this to the fluorescence intensity recorded fromcalibration beads included in the array.

[0073] The estimated number ofmolecules bound per bead is compared totheoretical binding curves derived from the law of mass action. Thethree curves shown correspond to values of the affinity constant, K_(A),of 10¹¹/molar, 10¹⁰/molar and 10⁹/molar, respectively. The initialnumber of antibodies per bead, R₀, equals 2×10⁵/bead and n_(B)=10⁵/ml.Each data point represents the average of three replicates, with anassay-to-assay variation of <45%. Setting the assay sensitivity tocorrespond to that level of fluorescence which yields a signal-to-noiselevel of unity in the assay images, the sensitivity of the cytokineassays characterized in FIG. 12 is set at ˜2,000 bound ligands/bead,corresponding to respective detected concentrations of 700 fM for TNF-αand 2 pM for IL-6.

[0074] While commercial ELISA kits use enzymatic amplification toenhance sensitivity, at the expense of additional complexity relating toassay conditions and controls, our bead array assay format, even withoutenzymatic amplification, our on-chip assay format permits monitoring ofcytokines at circulating levels (Normal TNF-α level in serum is 50-280fM and normal IL-6 level in serum is 0-750 fM.www.apbiotech.com/technical/technical_index.html), providing a dynamicrange which approaches that of standard, i.e. amplified single-analyteELISA assays (Assay kits of R & D Systems and Amersham (not the recentHigh-Sensitivity assays). Further improvements at hardware and softwarelevels are possible.

Example 7 Genotyping by Polymorphism Analysis

[0075] To illustrate the application of the present invention to theimplementation of genotyping, FIG. 13 shows the design of the assay inwhich five pairs of 20-mer binding agents corresponding to fourpolymorphic regions of a gene were coupled to color-encoded beads. Thepairs of binding agents α1, α2 and β1, β2 each display a singlenucleotide difference in their respective sequences; the pair 63, 64displays a difference of three nucleotides, the binding agents in theset γ1, γ3, γ3, γ4 display small insertions and deletions. The tenbinding agents were are divided into two subgroups of five which wereincorporated into two subarrays. In this example, there are severalhundred beads for each type. Following bead immobilization, an on-chiphybridization reaction was performed in TMAC buffer (2.25 Mtetramethylammonium chloride, 37 mM Tris pH 8.0, 3 mM EDTA pH 8.0, and0.15% SDS) at 55° C. for 30 min. The analyte is a 254-base PCR fragmentproduced from a patient sample and fluorescently labeled at the 5′-primeend with BODIPY 630/650 (Molecular Probes, Eugene, Oreg.). Imageacquisition was performed after replacing the assay buffer with freshTMAC buffer.

[0076]FIG. 14 shows decoding and assay images for one subarray. Eachbead shown in the assay image obtained after hybridization is analyzedto determine fluorescence intensity and bead type; as with the cytokineassay, the latter operation compares assay and decoding images using atemplate matching algorithm. FIG. 15 displays the resulting intensityhistograms for each bead type: in these histogram plots, the horizontalaxis refers to relative signal intensity from 0 to 1 and the verticalaxises refer to bead numbers. The histograms show that most of the beadsdisplaying probe α1 bind no analyte while most of the beads displayingprobe α2 exhibit significant binding; the mean signal level of α2-beadsexceeds that of α2-beads by a factor of 3.2, indicating that analytecontains DNA sequences complementary to α2 but not α1. For the patientsample presented here, the histogram indicates a genotype of the analyteDNA characterized by complementarity to binding agents α2, α2, γ3, γ4and δ4 in the polymorphic region of the gene.

Example 8 Gene Expression Analysis: cDNA Fragments

[0077] The method of the present invention has been used to fabricatearrays composed of beads displaying oligonucleotides as well as DNAfragments (e.g., up to 1,000 bases in length). Strands were biotinylatedat multiple positions by nick-translation and were attached tostreptavidin-functionalized beads (M-280, Dynal, Oslo, NO). Arrays wereformed using an AC voltage of 800 Hz at 10V_(pp).

Example 9 Looped Probe Design for Universal Labeling

[0078] A looped probe design in FIG. 16 takes advantage of fluorescenceenergy transfer to obviate the need for labeled target. As with themolecular beacon design (S. Tyagi, D. P. Bratu. F. R. Kramer, NatureBiotech. 16, 49-53 (1998)), the probe in FIG. 16 assumes two differentstates of fluorescence in the closed loop and open loop configurations,but in contrast to the molecular beacon contains a portion of itsbinding motif within the stem structure to permit molecular control ofstringency in competitive hybridization assays.

Example 10 Quantitative Multiplexed Profiling of Cytokine Expression

[0079]FIG. 17 displays a pair of assay and decoding images recorded froma single random array in a multiplexed sandwich immunoassay. An arraycontaining five distinct types of beads, each displaying a monoclonalanti-cytokine antibody (mAb), was exposed to a sample solution (such asserum) containing two cytokine antigens (Ag). Subsequent addition ofCy5.5-labeled secondary antibodies (pAb*) results in the formation ofternary complexes, mAb-Ag-pAb*. The on-chip immunoassay was performed byadding 300 μl of sample with 7 nM cytokines in assay buffer (20 mM NaPipH 7.4, 150 mM NaCl, 10 mg/ml BSA) to the bead array immobilized on thechip, and allowing the reaction to proceed at 37° C. for one hour. Thebuffer was replaced by adding 12 nM solution of labeled secondaryantibodies in assay buffer. After one hour of incubation at 37° C.,fresh buffer was added on top of the chip and image acquisition wasperformed. Antibodies and antigens used in the assays were obtained fromR&D Systems (Minneapolis, Minn.); the secondary antibody was labeledwith Cy5.5 using a standard kit (Amersham Pharmacia Biotech, Piscataway,N.J.).

[0080] The decoding image FIG. 17B shows five types of beads in afalse-color display with the same encoding pattern as that of FIG. 10.All beads are of the same size (5.5 μm diameter); the apparentdifference in the size of beads of different types in the decoding imageis an artifact reflecting different internal bead staining levels and“blooming” during CCD recording of the decoding image. Comparison (usingthe image analysis methods disclosed herein) of the decoding image withthe assay image in FIG. 14A reveals that active beads, of yellow andbright green types, captured TNF-α and IL-6, respectively. This assayprotocol has been extended to the following set of twelve cytokines:IL-1α, IL-1β, IL-2, IL-4, IL-6, TGF-β1, IL-12, EGF, GM-CSF, M-CSF, MCP-1and TNF-α. The on-chip immunoassay requires no additional washing otherthan changing reagent solutions between assay steps. Comparison betweenassay and decoding images shows that two different cytokines werepresent in the sample, namely IL-6 and TNF-α. The pre-formed arraysdescribed in this example also permit the determination of affinityconstants in a manner analogous to the analysis described in Example 6.

Example 11 Aptamers for Protein Profiling

[0081] Aptamers may be selected from large combinatorial libraries fortheir high binding affinities to serum proteins (L. Gold, B. Polisky, O.Uhlenbeck, M. Yarus, Annu. Rev. Biochem. 64: 763-797. (1995)). Randomencoded arrays of aptamer-functionalized beads would serve to monitorlevels of serum proteins; correlations in binding patterns on the array(see also Example 10) may serve as a phenotype of disease states.

Example 12 Mixed DNA -Protein Arrays

[0082] Of significant interest to genomic functional analysis is thefact that the method of the present invention accommodates protein andDNA arrays without change in array manufacturing methodology.Specifically, mixed arrays composed of beads displaying DNA andcorresponding proteins can be used to analyze the gene and gene productwithin the same fluid sample.

[0083] This has been demonstrated for a combination of immunoassay andDNA hybridization. For example, a mixed array composed of beadsfunctionalized with anti-cytokine monoclonal antibodies (mAb) and witholigonucleotides was produced. Two sequential assays were performed onthis single chip. First, an immunoassay was performed in accordance withthe protocol described in Example 10. Following completion of theon-chip immunoassay, image is acquired and the DNA analyte was added tothe hybridization buffer (2× SSC, 1× Denhardt's) at a finalconcentration of 20 nM and allowed to react at 37° C. for 1 hr. Freshhybridization buffer was added to the chip and image acquisition wasperformed to record of the additional hybridization assay.

Example 13 Affinity Fingerprinting

[0084] The analysis of receptor-ligand interactions relevant to priorart methods assumes ideal specificity. That is, only the ideal situationis considered of a single ligand present in solution reacting with itsmatching receptor and vice versa. However, in most multiple assaysystems, a considerable level of cross-reactivity may exist. That is,any single ligand may associate with several receptors, while any singletype of receptor may have a finite affinity towards more than oneligand.

[0085] The present invention includes a model that is developed toanalyze multiplexed READ assays for such a system under the followingassumptions: each of these reversible reactions is characterized by itsown affinity constant; no reaction occurs between the bulk species;there is no interaction between the complexes formed on the surface.These assumptions can be relaxed, at the expense of increasing thecomplexity of modeling, by accounting for reactions in the bulk andbetween the surface species. The standard reaction-diffusion equationfor single receptor-ligand pair formation [R. W. Glaser, Anal. Biochem.213, 152-161 (1993)], is generalized to allow for multiple reactions ateach bead surface: $\begin{matrix}{{\frac{\partial\left\lbrack {L_{i} \cdot R_{j}} \right\rbrack}{\partial t} = {{{k_{{on},{ij}}\left\lbrack L_{i} \right\rbrack}\left( {\left\lbrack R_{j,0} \right\rbrack - {\sum\limits_{n,m}^{\quad}\quad \left\lbrack {L_{m} \cdot R_{n}} \right\rbrack}} \right)} - {{k_{{off},{ij}}\left\lbrack {L_{i} \cdot R_{j}} \right\rbrack}\quad {\forall i}}}},j,{L_{i} \equiv {L_{i}\left( {t,x,0} \right)}}} & (2)\end{matrix}$

[0086] The first term on the right of Eq. (1) describes the associationof ligands and receptors into complexes and involves of concentration offree sites on the surface. The second term describes the disassociationof complexes by way of release of ligands, thereby freeing up receptorsites for further reaction. Since a maximum of (i×j) bimolecularcomplexes can form, there could be as many boundary conditions generatedfrom the above equation. For the equilibrium case, the left hand-side ofEq. (1) is set to zero, and the matrix of coaffinities, [K_(ij)]=k_(on,ij)/k_(off,ij), can then be defined to accommodatecross-reactivities between multiple species in the bulk and on thesurface. In a batch reactor under equilibrium conditions, we may solvethe system of differential equations to obtain the number of moleculesof each ligand bound on beads of each type. L₁ Ligand concentration  10pM L₂ Ligand concentration 100 pM R₀₁ Initial receptor concentration 1 ×10⁴/bead R₀₂ Initial receptor concentration 1 × 10⁴/bead n_(B1) Beadnumber density 1 × 10⁴/ml n_(B2) Bead number density 1 × 10⁴/ml [K]Coaffinity matrix [1 × 10¹¹ 1 × 10⁹ 1 × 10⁸ 1 × 10¹¹] 1/mole

[0087] As an illustrative example, the ligand distribution has beencalculated (from the model in Eq (1)) for a reference set of two ligandsand two types of receptors immobilized on two different sets of beads.The coaffinity matrix is assumed known for each ligand-receptorcombination in the reference set; to investigate the detection of athird ligand, it is assumed here that diagonal elements of the 2×2matrix, [K_(ij)], are large compared to off-diagonal elements. Thepresence of a third ligand in the reactor alongside the two originalligands perturbs the equilibria between the various complexes and thereactants in the reference system, and for ligand molecules tagged withfluorescent labels, the intensity observed from the perturbed systemdiffers from that observed in the reference case.

[0088]FIG. 18A shows the reference case in which the concentrations andcoaffinity matrix were set to the values shown in the accompanyingtable; the bead intensity was defined on a linear scale of 0-255, thelatter representing the intensity of the brightest beads. FIG. 18A showsthe contribution of each ligand to the bead intensity. Due to the lowerconcentration of L₁, the intensity of Bead 1 is less compared to Bead 2,cross-reactivities are essentially undetectable.

[0089] Next, the system was perturbed with a third ligand, taking theconcentration L₃ to be 1 pM and assuming that the new ligand hasconsiderable amount of cross-reactivity with each of the receptors;K_(3,1)=1×10¹¹ /M, K_(3,2)=1×10¹⁰ /M. Calculation of the fluorescentintensity of each bead in the presence of the third ligand yields thepattern in FIG. 18A which reveals an increase in the intensity of Bead 1due to the third ligand, while leaving the intensity of Bead 2unaffected due to the higher concentration of L₂ in the system and thelower affinity of L₃ to R₂. Thus, L₃ may be detected under the conditionthat it has a relatively high affinity to one of the receptors and is insignificant amount compared to the competing ligand.

[0090] The evaluation of the coaffinity matrix (and comparison withtheoretical modeling as disclosed herein) under conditions in which amixture of ligands is permitted to interact with a multiplicity ofreceptors arranged in a random encoded bead array format provides amethodology to establish a characteristic feature set ofcross-correlations in the mutual competitive binding affinities ofmultiple ligands and receptors. These co-affinities provide a robustmeans to characterize receptor-ligand binding equilibria by theiraffinity fingerprinting patterns. Deviations from well-defined referencecases also permit detection of “perturbing” ligands in solutions.

Example 14 Multiplexed Analysis of Reaction Kinetics

[0091] As illustrated in the foregoing examples, extensive washinggenerally is not required to discriminate beads from a background ofsolution fluorescence. Consequently, assay image sequences may berecorded in a homogeneous assay format to document the evolution of abinding reaction and to determine kinetic data for each of the bindingreactions occurring.

[0092] Homogeneous binding assays may be performed in simple “sandwich”fluidic cartridges permitting optical microscopic imaging of the beadarray and permitting the introduction of an analyte solution into achamber containing a random encoded array of beads. More generally, thearray also may be exposed to an analyte or other reaction mixtures underconditions of controlled injection of fluid aliquots or continuous flowof reactants or buffer. Using theoretical modeling, optimal combinationsof relevant performance control parameters of this bead array reactormay be identified to minimize the time to equilibration or to maximizethe portion of analyte captured by the array [K. Podual and M. Seul, TMKP-99/02]. Flow rate can be controlled by any of a number of availablepumping mechanisms [M. Freemantle, C&EN, 77: 27-36]. TABLE List ofparameters used in simulations (FIG. 18) Parameter, units Value InitialReceptor Coverage c_(R,0), moles/m² 8 × 10⁻⁹ Vol Flow Rate, Q, μl/s  1.0Diffusivity, D, cm²/s 1 × 10⁻⁷ ON-Rate, k_(on), /(M s) 1 × 10⁵ AffinityConstant, K_(A), /M 1 × 10¹¹ “Sandwich” Reactor Gap Size H, mm  0.1Reactor Length, L, mm 10 Reactor Width, W, mm 10

[0093] The analysis of image sequences permits kinetic data to begenerated from which ON-rates and OFF-rates are determined with the aidof a theoretical model of the reaction-diffusion kinetics of the typeillustrated in the foregoing example in FIG. 19. FIG. 19A displaysstages in an adsorption-desorption cycle involving solution-borneanalytes and a bead array immobilized at the bottom of a “sandwich”reaction chamber. The first panel depicts the initiation of theadsorption process; the second panel depicts the state of the reactorclose to equilibrium when most of the beads have reached equilibrium;the last panel depicts the state of the reactor under the desorptioncycle in which ligand-free fluid is injected and adsorbed moleculesdesorb from the bead surface. FIG. 19B displays theadsorption-desorption kinetics of a single receptor-single ligand systemobtained by numerical solution of a reaction-diffusion system for asingle type of receptor-ligand reaction; two cases of differentconcentrations of ligand are shown. Parameters used in the simulationare listed in the accompanying Table.

[0094] In contrast to prior art methods [D. G. Myszka, Curr. Opin.Biotechnol. 8: 50-57.], the present method relies on imaging and permitsmultiplexing. In addition, generalized models of the type introduced inExample 6 permit the analysis of complex binding kinetics for multiplesimultaneous receptor-ligand interactions even in the presence ofcross-reactions between multiple ligands and receptors.

[0095] The ability to monitor reaction kinetics in an array format willenable several approaches to enhancing the specificity ofreceptor-ligand or binding agent-analyte interactions in complexmixtures. For example, temperature programming may be invoked to enhancethe specificity of DNA hybridization reactions. Similarly, thestringency of conditions applied to a hybridization reaction may bevaried while the array response is being monitored; for example,hybridization may be conducted in a hybridization buffer underconditions leading to excess “non-specific” binding; specificity isenhanced by switching to a wash buffer of increasing stringency whilemonitoring the array response.

Example 15 Multi-Step Assay Sequences Using Encoded Arrays of MagneticParticles

[0096] Methods and apparatus using biochemically functionalizedsuper-paramagnetic particles for sample preparation in molecular andcellular biology and for a variety of enzyme-catalyzed on-bead reactionshave been described [“Biomagnetic Techniques in Molecular Biology”,Technical Handbook, 3^(rd) Edition, 1998, Dynal, Oslo, NO). Thesebead-based methods can be combined with the Random Encoded ArrayDetection format of the present invention to implement multi-stepon-chip assay manipulations.

[0097] For example, FIG. 20 illustrates the integration of a sequence ofsteps in a miniaturized format for multiplexed genotyping using a singlechip with multiple compartments. First, cells are captured from apatient sample by affinity selection using functionalized magneticbeads, cells are lysed electrically or chemically in a firstcompartment, and genomic DNA is captured to the surface of amultiplicity of magnetic beads by non-specific binding; next, beads arecollected by magnetic force into a second compartment which is influidic contact with the first compartment, within which the beads andDNA are washed with desired buffers; next, beads are further transferredto a location where PCR is performed using bead-coupled DNA as atemplate; multiple PCR strategies known in the art are available forthis step [F. Fellmann, et.al., Biotechniques, 21:766-770]; next, PCRproducts released into are captured by hybridization to a pre-assembledrandom encoded array displaying binding agents that are specific todifferent polymorphisms targeted by the PCR amplification.

[0098] The use of encoded magnetic particles in conjunction with theoptical programmability of LEAPS confers the ability to form reversiblyimmobilized arrays and to conduct programmable multi-step assaysequences under conditions in which beads are used in suspension whenthis is most favorable, for example to enhance reaction kinetics, andarrays are formed in real-time when this is most favorable, for exampleto provide a highly parallel format for imaging detection and assayread-out.

[0099] For example, as illustrated in FIG. 21, the following sequence ofsteps could be integrated in a miniaturized format for the formation ofa cDNA bead array. First, a pool of encoded magnetic beads, each beadtype displaying a gene-specific probe, is introduced to an mRNA pool,and mRNA molecules are hybridized to their corresponding beads; next,on-bead reverse transcription (RT) is performed using bead-attached mRNAas template [E. Horenes, L. Korsnes, US 005759820]; next mRNA isreleased from the beads; next beads are directed to the surface of acustom-designed chip and a cDNA bead array is formed using LEAPS. Suchan array could serve to display binding agents in a gene profilingexperiment using another set of mRNA as the target. Alternatively, thecDNA array could be analyzed for its own expression by applying a poolof labeled DNA binding agents to profile the genes of interest withinthe array.

Example 16 Synthesis of Super-paramagnetic Iron Oxide γ-Fe₂O₃(maghemite) Particles

[0100] The synthesis was carried out in reversed micellar solutionscomposed of the anionic surfactant, bis(2-ethylhexyl)sodiumsulfosuccinate (AOT) and isooctane (Kommareddi et al., Chem. Mater.1996, 8, 801-809)obtained from Aldrich Chemical Co., Milwaukee, Wis.Stock solutions of 0.5M AOT were used in preparing the reversed micellarsolutions containing the reactants FeSO₄ (Sigma Chemical Co., St. Louis,Mo.) and NH₄OH (Sigma Chemical Co., St. Louis, Mo.). Specifically, 0.45ml of 0.9M FeSO₄ was added to 5 ml of 0.5M AOT in isooctane, separately0.45 ml of NH₄OH was added to 5 ml of 0.5M AOT in isooctane. Thereaction was initiated by adding the NH₄OH reversed micellar solution tothe FeSO₄ reversed micellar solution under vigorous stirring. Thereaction was allowed to proceed for 2-3hrs and then the solvent wasevaporated at 40° C. to obtain a dry surfactant iron oxide composite.This composite was re-dispersed in the organic solvent of choice to givea deep red colored transparent solution.

Example 17 Synthesis of Fluorescently Colored and Magnetic Polymer BeadComposites

[0101] A stock solution of hydrophobic fluorescent dye and the ironoxide particles was made by re-dispersing the dried magnetic compositeand the dye in the solvent of choice, for example a CHCl₃ (AldrichChemical Co., Milwaukee, Wis.) or CH₂Cl₂/CH₃OH mixture (70/30 (v/v))(Aldrich Chemical Co., Milwaukee, Wis.). A predetermined amount ofpolymer beads was washed thoroughly in methanol (3×) and then evaporateddry. Simultaneous incorporation of the fluorescent dye and the ironoxide nanoparticle was achieved by swelling the beads in organicsolvent/nanoparticle/dye mixture. The swelling process was completedwithin 1 hr. Following this the polymer beads were separated bycentrifugation and washed with methanol(3×) followed by isooctane(2×)and then methanol(2×) and finally redispersed in 0.2% SDS -DI watersolution.

What is claimed is:
 1. A method of determining elements of a co-affinity matrix which describes pair-wise analyte-binding interactions in a competitive multiconstituent equilibrium reaction comprising: providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate; determining the identity of said binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith; contacting the binding agents with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex; detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed; and determining the identity of the binding agents involved in the complex formation by the chemically or physically distinguishable characteristic associated with the corresponding particles, wherein said change in optical signature for each type of the analyte-binding agent complexes determines affinities characterizing said analyte-binding agent interaction and said affinities providing elements of a co-affinity matrix which describes pairwise analyte-binding agent interactions in a competitive multiconstituent equilibrium reaction.
 2. A method of determining elements of a co-affinity matrix which describes pair-wise analyte-binding interactions in a competitive multiconstituent equilibrium reaction comprising: providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, contacting the binding agents with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex; forming a planar array of the particles on a substrate; detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed; and determining the identity of the binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith, the determining step occurring either before or after the detecting step, and wherein said change in optical signature for each type of the analyte-binding agent complexes determines affinities characterizing said analyte-binding agent interaction and said affinities providing elements of a co-affinity matrix which describes pairwise analyte-binding agent interactions in a competitive multiconstituent equilibrium reaction.
 3. The method of claim 1 or 2, wherein the co-affinity matrix obtained is used to characterize the analyte.
 4. The method of claim 3, wherein a combination of the coefficients of the co-affinity matrix and known concentrations of analyte and binding agents participating in the formation of analyte-binding agent complexes serves to define a competitive binding interaction descriptor.
 5. The method of claim 1 or 2, further comprising the determination of the affinity constant for each type of the analyte-binding agent interaction, wherein each affinity constant is calculated by determining the number of analyte-binding agent complexes formed from said change in the optical signature of each type of the analyte-binding agent complexes formed.
 6. A method of determining the presence or absence of an impurity in a sample comprising: preparing a co-affinity matrix of a reference analyte with a set of binding agents according to the method of claim 1 or 2, and recording said co-affinity matrix, introducing a sample containing said reference analyte and possibly an impurity analyte, and determining the co-affinity matrix subsequent to the addition of the sample; and detecting for the deviation in the coefficients of the co-affinity matrix of the reference analyte subsequent to the addition of the sample that might contain an impurity, wherein the deviation in the co-affinity matrix indicates the presence of an impurity in said sample.
 7. A method of analyzing the kinetics of molecular binding interactions governing the association of analyte-binding agent complexes formed between one or more analytes and one or more binding agents, comprising: providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate; determining the identity of said binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith; contacting, at a preset initial time, the binding agents in the array with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex; detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed at preset intervals following said preset initial time; and determining, for each type of analyte-binding agent complex, the association rate constants from the time dependence of said changes in optical signature associated with said particles.
 8. A method of analyzing the kinetics ofmolecular binding interactions governing the dis-association of analyte-binding agent complexes formed between one or more analytes and one or more binding agents, comprising: providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate; determining the identity of said binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith; contacting the binding agents in the array with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex; detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed; exchanging, at a preset exchange time, the analyte solution with a second solution containing no analyte to allow the bound analytes to unbind from the analyte-binding agent complexes, said unbinding resulting in a change in the optical signature associated with the particles having analyte-binding agent complexes, and recording, at preset intervals following said preset exchange time, said changes in optical signature and deterining, for each type of analyte-binding agent complex, the dis-association rate constants from the time dependence of said changes in optical signature associated with said particles.
 9. The method of any of claims 1 to 8, wherein the change in the optical signature comprises a change in the fluorescent intensity associated with the particles involved in the binding interaction.
 10. The method of any one of claims 1 to 8, wherein the determining of the identity of the binding agents in the array comprises taking a decoding image of the array that detects the chemically or physically distinguishable characteristic of each bead in the array, and wherein the optical signature detecting step comprises taking an assay image of the array that detects the change in the optical signature associated with the beads.
 11. The method of claim 10, wherein the assay image and the decoding image are compared using a template matching algorithm.
 12. The method of any one of claims 1 to 8, wherein the planar particle array is immobilized on the substrate.
 13. The method of any one of claims 1 to 8, wherein the particles are associated with a chemically distinguishable characteristic.
 14. The method of claim 13, wherein the chemically distinguishable tag comprises a fluorophore tag.
 15. A method of performing a bioassay comprising: providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate; generating a de-coding image of the array showing the location of each binding agent in the array; contacting the binding agents with a sample that may contain an analyte so as to allow the analyte, if present in the sample, to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a corresponding or a proportional change in the optical signature associated with the particles whose binding agent is involved in the formation of the complex; generating an assay image of the array which detects the change in the optical signature associated with said particles, and deriving from the change in the optical signature the number of analyte-binding agent complexes formed; and determining the identity of the analyte in the analyte-binding agent complex by comparing the decoding image with the assay image.
 16. A method of performing a bioassay comprising: providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, contacting the binding agents with a sample that may contain an analyte so as to allow the analyte, if present in the sample, to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a corresponding or proportional change in the optical signature associated with the particles whose binding agent is involved in the formation of the complex; forming a planar array of the particles on a substrate; generating an assay image of the array which detects the change in the optical signature associated with said particles, and deriving from the change in the optical signature the number of analyte-binding agent complexes formed; generating a decoding image of the array showing the location of each binding agent in the array, wherein the decoding image is generated either before or after generating the assay image; and determining the identity of the analyte in the analyte-binding agent complex formed by comparing the decoding image with the assay image.
 17. The method of claim 15 or 16, wherein the comparing of the assay and the decoding image comprises use of optical microscopy apparatus including an imaging detector and computerized image capture and analysis apparatus.
 18. The method of claim 15 or 16, wherein the analyte comprises a ligand and the binding agents comprise receptors.
 19. The method of claim 15 or 16, wherein the analyte and the binding agents comprise nucleotide fragments, and wherein the number of hybridization complexes formed between the nucleotide fragment and its complementary sequence fragments is determined.
 20. The method of claim 15 or 16, wherein the particles comprise magnetic particles distinguishable by a chemical or physical characteristic that uniquely identifies the binding agent attached thereto.
 21. The method of claim 20, wherein the chemical or physical characteristic comprises one or more fluorophore dyes spectrally distinguishable by excitation wavelength, emission wavelength, excited-lifetime or emission intensity.
 22. The method of claim 21, wherein the array of magnetic particles is assembled by application of a magnetic field to said particles.
 23. The method of claim 15 or 16, further comprising calculating the affinity constant of a binding interaction between an analyte and its binding agent from the number of the analyte-binding agent complexes formed.
 24. A method of integrating sample preparation and bioassay using magnetic particles comprising: providing a plurality of magnetic particles comprising at least two different particle populations, each population being distinguishable by a recognition molecule attached thereto, wherein the particles are attached to a chemical characteristic that uniquely identifies a biomolecule of interest that selectively binds to the recognition molecule; providing a biological fluid containing biomolecules and allowing said biomolecules to interact with the recognition molecules on the magnetic particles; removing the fluid along with unbound components thereof; transforming the biomolecules bound to the magnetic particles to produce transformed biomolecules, wherein the transformed biomolecules remain attached to the magnetic particles on which they are synthesized; performing a bioassay according to the method of claim 15 or 16, wherein the binding agents comprise the transformed biomolecules.
 25. The method of claim 24, wherein the biomolecules of interest comprises mRNA and the transforming comprises reverse transcribing said mRNA to produce cDNA, which is attached to the magnetic particles.
 26. The method of claim 24, wherein the sample preparation and bioassay occur in the same compartment.
 27. A method for performing a bioassay involving integration of sample preparation and parallel molecular interaction assay analysis, comprising providing an apparatus comprising at least a sample preparation compartment and an assay compartment, and means for fluidically connecting the sample and the assay compartments; providing, in the sample preparation compartment, a biological fluid containing a biomolecule of interest and a plurality of magnetic particles capable of binding to the biomolecule of interest, and allowing the magnetic particles to bind the biomolecules of interest; removing the biological fluid along with unbound components of said fluid, while retaining the magnetic particles and the biomolecules bound to said particles; releasing said biomolecules from said magnetic particles and transporting said biomolecules from the sample preparation compartment to the assay compartment through the fluidic means; and performing a bioassay according to the method of 15 or 16, wherein the analyte in the bioassay comprises transported biomolecules of interest.
 28. The method of claim 27, further comprising transforming the biomolecule of interest, which is then used as an analyte in the bioassay.
 29. The method of claim 27, wherein the biomolecule of interest comprises mRNA and the transformation comprises reverse transcription of said mRNA to produce cDNA, and wherein the analyte in the bioassay comprises said cDNA and the binding agents comprises oligonucleotides or other DNA probes.
 30. The method of claim 29, wherein the reverse transcription occurs in the sample preparation compartment, while said mRNA is bound to the magnetic particles, and the cDNA is released from said magnetic particles after the reverse transcription and transported to the assay compartment and used as an analyte in the bioassay.
 31. A method of preparing monodisperse magnetic fluorescent particles comprising providing polymeric microparticles and swelling said microparticles in an organic solvent containing one or more and magnetic nanoparticles to produce magnetic particles, wherein the fluorescent dyes and the magnetic nanoparticles are distributed throughout the magnetic particles without being localized at specific locations with the particle.
 32. An array comprising a plurality of magnetic particles comprising at least two different particle populations distinguishable by a chemical characteristic associated therewtih, wherein the magnetic particles are assembled on a planar array in a compositionally random manner.
 33. The method of claim 5, wherein the analyte specifically binds to one binding agent but not to other binding agent(s) present.
 34. The method of claim 33, wherein, using the law of mass action, affinity constant K is determined from said change in optical signature following conversion to yield [LR], given [L₀], [R₀], and n_(B), where [L₀] is the initial analyte concentration, [R₀] is the number of binding agents per bead and n_(B) is the number of beads.
 35. The method of claim 33, wherein, using the law of mass action, affinity constant K is determined from said change in optical signature following conversion to yield [LR], for at least two measurements involving different bead numbers, n_(B) and given initial analyte concentration [L₀].
 36. The method of claim 33, wherein, using the law of mass action, affinity constant K is determined from said change in optical signature following conversion to yield [LR], for at least two measurements involving different number of binding agents per bead, [R₀] and given initial analyte concentration [L₀].
 37. The method of claim 33, wherein, using the law of mass action, concentration of analyte [L], is determined from said change in optical signature following conversion to yield [LR], given K, n_(B) and [R₀]
 38. The method of claim 33, wherein in a two-step assay involving the use of a first labeled analyte of interest in the first binding reaction, followed by injection of a second analyte with known affinity constant K′, the affinity constant K is determined from said the optical signature before and after injection of the second analyte, following conversion to yield [LR]₁ and [LR]₂ given the initial concentrations of the analytes, [L₀] and [L₀]′ and [R₀].
 39. The method of claim 33, wherein in a two-step assay involving the use of a first labeled analyte of interest in the first binding reaction, followed by injection of a second analyte with known affinity constant K′, the affinity constant K is determined from said the optical signature before and after injection of the second analyte, following conversion to yield [LR]₁ and [LR]₂ given the initial concentrations of the analytes, [L₀] and [L₀]′ and n_(B).
 40. The method of claim 33, wherein: using the law of mass action, the initial analyte concentration [L₀] is determined from said change in optical signature following conversion to yield [LR], given K, [R₀] and n_(B). 